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The expression and distribution patterns of the taste signaling molecules, α-gustducin (Gαgust) and α-transducin (Gαtran) G-protein subunits, were studied in the gastrointestinal tract of the chicken (Gallus domesticus) using the immunohistochemical method. Gαgust and Gαtran immunoreactive (-IR) cells were observed in the mucosal layer of all examined segments, except the esophagus, crop, and the saccus cranialis of the gizzard. The highest numbers of Gαgust and Gαtran-IR cells were found in the proventriculus glands and along the villi of the pyloric, duodenum, and rectal mucosa. Gαgust and Gαtran-IR cells located in the villi of the jejunum, ileum, and cloaca were much less numerous, while only a few Gαgust and Gαtran-IR cells were detected in the mucosa of the proventriculus and cecum. In the crypts, IR cells were observed in the small and large intestine as well as in the cloaca. Gαgust and Gαtran-IR cells displayed elongated ("bottle-" or "pear-like") or rounded shape. The demonstration of Gαgust and Gαtran expression provides evidence for taste receptor mediated mucosal chemosensitivity in the chicken gastrointestinal tract., (© 2016 Poultry Science Association Inc.)

During the transit in the female genital tract, spermatozoa are exposed to an environment that varies in composition from the vagina to the oviduct. Because G proteins, α-gustducin and α-transducin, are accepted as markers of chemosensitive cells, this study was aimed at assessing whether these proteins are expressed in boar germ cells. Ejaculated sperm extracts were analyzed by Western blot, and indirect immunofluorescence was performed on testis sections, smears of epididymal and ejaculated spermatozoa, sperm cells after in vitro induction of capacitation and acrosome reaction (IVAR), and in sperm cells bound to zona pellucida during IVF. Based on immunoblot results, both G proteins are present in boar sperm. In the testicular tissue sections, α-gustducin and α-transducin positivity was recorded in the germinal cells near the tubular lumen, whereas no positive signal was evident in spermatogonia located in the outer region of the seminiferous tubules. α-Gustducin expression in epididymal and ejaculated spermatozoa was mainly detectable in both the acrosome and the principal piece of the tail, whereas α-transducin was confined to the acrosome and the midpiece. No changes after in vitro induction of capacitation and IVAR were observed, except for the disappearance of acrosomal positivity in reacted spermatozoa. In sperm bound to zona pellucida, the G protein signal was congruent with that observed in IVAR cells. To the best of our knowledge, this is the first description of α-transducin in mammalian sperm and the first description of α-gustducin in boar sperm. Further studies are needed to clarify the possible role of these G proteins in sperm physiology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A routine use of boar-sexed semen is limited by the long sorting time necessary to obtain an adequate number of sexed spermatozoa for artificial insemination and by the high susceptibility of spermatozoa of this species to damages induced by sorting procedure and subsequent cryopreservation. The aim of this work was to study the impact of encapsulation in barium alginate membrane on sorted boar spermatozoa by evaluating membrane integrity, chlortetracycline staining patterns, protein tyrosine phosphorylation, and Hsp70 immunolocalization during storage over 72 hours in liquid or encapsulated form. The encapsulation procedure significantly (P < 0.05) decreased the overall membrane integrity of control unsorted semen (81.8 vs. 57.4, CTR vs. CPS), but did not negatively affect the overall viability and the chlortetracycline staining patterns of sorted encapsulated cells. Moreover, encapsulation significantly decreased (P < 0.05) the overall phosphotyrosin A pattern cell percentage in unsorted (98.4 vs. 92.6, CTR vs. CPS) but not in sorted semen (64.0 vs. 74.2; SORT CTR vs. SORT CPS). As for Hsp70, the overall percentage of cells displaying the different patterns was significantly influenced (P < 0.05) by treatment but not by storage time. The sorting procedure seems to induce the major changes, whereas encapsulation tends to exert a protective effect on sorted semen by increasing the percentage of spermatozoa displaying the T pattern (2.8 vs. 24.3; SORT CTR vs. SORT CPS). In conclusion, our data confirm that the damaging impact of the encapsulation in barium alginate capsules seems to be limited when compared with that of the sorting procedure and, moreover, the association of the two procedures does not result in an algebraic sum of the negative effects. These results suggest the possibility of a future utilization of the encapsulation technology in order to store sorted spermatozoa and permit their controlled release in the female genital tract., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although sorted semen is experimentally used for artificial, intrauterine, and intratubal insemination and in vitro fertilization, its commercial application in swine species is still far from a reality. This is because of the low sort rate and the large number of sperm required for routine artificial insemination in the pig, compared with other production animals, and the greater susceptibility of porcine spermatozoa to stress induced by the different sex sorting steps and the postsorting handling protocols. The encapsulation technology could overcome this limitation in vivo, protecting and allowing the slow release of low-dose sorted semen. The aim of this work was to evaluate the impact of the encapsulation process on viability, acrosome integrity, and on the in vitro fertilizing potential of sorted boar semen. Our results indicate that the encapsulation technique does not damage boar sorted semen; in fact, during a 72-hour storage, no differences were observed between liquid-stored sorted semen and encapsulated sorted semen in terms of plasma membrane (39.98 ± 14.38% vs. 44.32 ± 11.72%, respectively) and acrosome integrity (74.32 ± 12.17% vs. 66.07 ± 10.83%, respectively). Encapsulated sorted spermatozoa presented a lower penetration potential than nonencapsulated ones (47.02% vs. 24.57%, respectively, P < 0.0001), and a significant reduction of polyspermic fertilization (60.76% vs. 36.43%, respectively, polyspermic ova/total ova; P < 0.0001). However, no difference (P > 0.05) was observed in terms of total efficiency of fertilization expressed as normospermic oocytes/total oocytes (18.45% vs. 15.43% for sorted diluted and sorted encapsulated semen, respectively). The encapsulation could be an alternative method of storing of pig sex sorted spermatozoa and is potentially a promising technique in order to optimize the use of low dose of sexed spermatozoa in vivo., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Sperm sorting is a useful technology that permits sex preselection. It presents some troubles because of low fertility after the process. The main aim of this work was to analyze the putative existence of capacitation-like changes in both boar and bull sperm subjected to sex sorting that could lead to a detriment of semen quality. The parameters used were CTC staining patterns, actin cytoskeleton organization and tyrosine phosphorylation patterns; the last two were determined by both western blotting and immunofluorescence. Sex sorted spermatozoa were compared with fresh, in vitro capacitated and in vitro acrosome reacted sperm. In both species, sex sorted sperm showed a CTC staining pattern similar to that observed after in vitro capacitation. The actin pattern distribution after sperm sorting also tended to be similar to that observed after in vitro capacitation, but this effect was more pronounced in bull than in boar spermatozoa. However, actin expression analysis through western blot did not show any change in either species. The tyrosine phosphorylation pattern in boar sperm was practically unaltered after the sex sorting process, but in bulls about 40% of spermatozoa had a staining pattern indicative of capacitation. Additionally, western blotting analysis evidenced some differences in the expression of protein tyrosine phosphorylation among fresh, capacitated, acrosome reacted and sex sorted sperm cells in both species. Our results indicate that not all the sex-sorted-related modifications of the studied parameters were similar to those occurring after "in vitro" capacitation, thus suggesting that sex sorting-induced alterations of sperm function and structure do not necessarily indicate the achievement of the capacitated status of sorted sperm., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A routine use of boar sexed semen is far from being a reality due to many limiting factors among which is the long sorting time necessary to obtain the adequate number of sexed spermatozoa for artificial insemination and the high susceptibility to damages induced by cryopreservation. The aim of this study was to evaluate the modification induced by 24-26 h storage on sorted boar spermatozoa on the basis of their viability, acrosome status, Hsp70 presence, and in vitro fertilizing ability. The percentage of viable cells, according to SYBR green/PI staining, was negatively affected (P < 0.05) by sorting procedure. Moreover, liquid storage significantly (P < 0.05) reduced membrane integrity of sorted spermatozoa as compared to all the other groups. Neither sorting nor storage influenced the percentage of live cells with reacted acrosome, according to FITC-PNA/PI staining. Sorted samples, after 24-26 h storage, were characterized by an increase (P < 0.05) of sperm cells negative for Hsp70, as observed by immunofluorescence, and by a decrease (P < 0.05) in Hsp70 content, as evidenced by western blot. While sorting procedure did not adversely affect both penetration rate and total efficiency of fertilization, these parameters were negatively (P < 0.05) influenced by storage after sorting. In order to minimize damages that compromise fertility and function of sex-sorted boar spermatozoa, the mechanisms by which sorting and liquid storage cause these injures require further study., (Copyright 2010 Elsevier Inc. All rights reserved.)

Daidzein, an isoflavone abundant in soybeans and other legumes, displays estrogen like properties. This study was aimed at evaluating the effect of daidzein (1 and 10 microM) on nuclear and cytoplasmic maturation of pig oocytes and on steroidogenic activity of cumulus cells. Daidzein supplementation during IVM had no effect on nuclear maturation and on fertilization traits. By contrast, both concentrations significantly (P < 0.05) inhibited progesterone production by cumulus cells after 24 and 48 h of culture while they did not induce any effect on estradiol production. Furthermore, daidzein did not exert any effect on the percentage of embryos that developed to blastocyst stage, on the number of blastomeres per blastocyst, or on the level of Hsp-70 and -90 gene transcript. Overall, our data demonstrate that daidzein added during oocyte maturation does not affect pig embryo development even if it markedly inhibits progesterone production by cumulus cells. Further studies are needed to evaluate the possible effect of daidzein during embryonic development., (Copyright 2010 Elsevier Inc. All rights reserved.)